FIBRONECTIN BINDING PROTEIN; MONOCLONAL ANTIBODY AND THEIR USE IN PREVENTING BACTERIAL ADHESION

Abstract: A monoclonal antibody (Mab), or a fragment thereof, that binds to one or more epitopes of a matrix binding protein and an isolated D1-D4 polypeptide from a Staphylococcus aureus Fbp and their use in the prevention of adhesion of bacteria, in particular gram positive bacteria, to extracellular matrix proteins on in-dwelling devices or to matrix proteins in wounds.

Description: FIBRONECTIN BINDING PROTEIN; MONOCLONAL ANTIBODY AND THEIR USE IN PREVENTING BACTERIAL ADHESION. The present invention relates to novel polypeptides and monoclonal antibodies, their preparation and their use to combat infection at the site of wounds, 5 surgical implants and other in-dwelling devices such as catheters. The present invention also relates to isolated nucleic acids encoding the polypeptide and to recombinant host cells transformed with DNA encoding the polypeptide. One of die major complications associated with the clinical use of implanted materials and in-dwelling devices is bacterial infection. In particular, staphylococci 10 have frequently been implicated in medical device-related infections (Dankeit et al 1986, CRC Rev Biocompatability 2, 219-301). Once established the infection is virtually impossible to treat resulting in implant failure. It has been suggested that the adhesion of a microorganism to a surface is an initial step in the development of such infections (Quie and Belani, 1987, J. Infec. 15 Dis. 156: 543-547 ) and there is now evidence to suggest that a specific adhesion mechanism is involved in the pathogenesis of foreign body infections (Vaudaux et al. 1990, J. BiomaL Appl.5: 134-153 ). Soon after coming into contact witfi blood, inert materials, such as used for intravenous cannulae and prosthetic implants, are almost immediately coated with a layer of extracellular matrix proteins, (Cottanaro et al 20 1981 , Transactions of the American Society for Artificial Internal Organs 27: 391-395 ). In particular this layer includes the plasma protein lϊbronectin and it is believed that staphylococci are able to bind to fibronectin through bacterial cell surface receptor proteins known as fibronectin binding protein (Fbp). However , some studies have suggested that blood proteins do not promote adherence of 25 staphylococci to biomaterial (eg. Muller et al 1991, IiifecLlmmun. 59: 3323-3326) thereby discouraging research into the interaction of these bacteria with these proteins as an approach in the prevention of adhesion to biomaterials. Fibronectin binding proteins have been isolated from Staphylococcus aureus and the nucleotide sequence subsequently established [ Signas, C. et al. (1989) Proc. 30 Nat Acad. Sci 86, 99-703 ; Jonsson, K. et al. (1991) Eur. J. Biochem. 202, 1041 - 1048] (Fbp A and FbpB respectively). The primary fibronectin binding domain of this protein has been identified as a homologous unit (usually of 38 amino acids) that is repeated three times (D1-D3 region) and partially repeated a fourth time (D4 region). 35 Previous attempts to combat staphylococcal adhesion to implants have involved modification of the surface of the prosthetic material to discourage adhesion of proteins ; e.g. coating widi a "non-stick" material such as PTFE, or bonding antibiotics to the surface (Kamal et al., 1991, J. Amer. Med. Assoc.265, 2364-2368 ). There have also been proposals to use non-steroidal anti-inflammatory drugs to prevent adhesion of staphylococci to medical polymers (Farber and Wolff 1992, J. Infect. Dis. 166: 861-865). EP0163623, EP0294349, EP0397633 and WO92 02555 disclose certain fibronectin binding polypeptides from S. aureus. One approach adopted in ϊhe present invention is to use a monoclonal antibody that binds to matrix binding proteins, such as fibronectin binding protein, to block the adhesion of bacteria to matrix proteins. Accordingly, in one broad aspect the present invention is directed to the use of a monoclonal antibody (Mab), or a fragment thereof, that binds to one or more epitopes of a matrix binding protein in the prevention of adhesion of bacteria, in particular gram positive bacteria, to extracellular matrix proteins on in-dwelling devices or to extracellular matrix proteins in wounds. The invention particularly relates to the manufacture of a medicament for such uses. The effect of the Mab or fragment is to block the site on the matrix binding protein that is associated with binding to d e matrix protein. In-dwelling devices include surgical implants, prosthetic devices and catheters, i.e., devices that are introduced to the body of a patient and remain in position for an extended time. Such devices include, for example, artificial joints, heart valves, pacemakers, vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinary catheters, continuous ambulatory peritoneal dialysis (CAPD) catheters, etc. The invention is particularly concerned widi the use of monoclonal antibodies that will prevent the adhesion of staphylococci such as S. aureus and coagulase- negative staphylococci, such as S. epidermidis to such indwelling devices. Accordingly the monoclonal antibody is preferably directed against epitopes of matrix binding proteins derived from such bacteria. Preferably the matrix binding protein to be recognised by the monoclonal antibody is fibronectin binding protein. In fibronectin binding protein it is known that a region identified as the D1-D4 domain is especially relevant in binding to fibronectin (Signas. C et al. 1989 op. cit). Accordingly, in a preferred embodiment the monoclonal antibody is directed against an epitope present on the D1-D4 domain of fibronectin binding protein or a component unit diereof. Novel monoclonal antibodies described above and their fragments form a further aspect of the invention. The antibody may be eidier intact antibody of M r approx 150,000 or a derivative of it, for example a Fab fragment or a Fv fragment as described in Skerra, A and Pluckthun, A (1988) Science 240 1038-1040. If two antigen binding domains are present each domain may be directed against a different epitope - termed 'bispecifϊc' antibodies. The antibody or derivative thereof may be prepared by conventional means for example by established monoclonal antibody technology (Kohler, G. and Milstein, C. (1975) , Nature, 256, 495-497) or using recombinant means e.g. combinatorial libraries, for example as described in Huse, W.D. et al., (1989) Science 246,1275- 1281. Preferably the antibody or derivative is prepared by expression of a DNA polymer encoding said antibody in an appropriate expression system. The choice of vector for the expression system will be determined in part by the host, which may be a prokaryotic cell, such as E. coli (preferably strain B) or Streptomyces sp. or a eukaryotic cell, such as a mouse C127, mouse myeloma, human HeLa, Chinese hamster ovary, filamentous or unicellular fungi or insect cell. The host may also be a transgenic animal or a transgenic plant [for example as described in Hiatt,A et α/.,(1989) Nature 34, 76-78]. Suitable vectors include plasmids, bacteriophages, cosmids and recombinant viruses, derived from, for example, baculoviruses and vaccinia. The Fab fragment may also be prepared from its parent monoclonal antibody by enzyme treatment, for example using papain to cleave the Fab portion from the Fc portion. The monoclonal antibody may be generated initially using as immunogen fibronectin binding protein, or the D1-D4 region of fibronectin binding protein. Fibronectin binding protein of Staphylococcus aureus is known to exist in at least two variants FbpA and FbpB [Jonsson et α/.(1991), op. cit.]. The binding domain of either of the above fibronectin binding proteins may be used as immunogen to generate a Mab of this invention. We believe we have now identified as a novel compound a new fibronectin binding protein from S. aureus J2385 (see Example 2 in particular). In particular we have isolated as a novel compound a polypeptide comprising essentially the D1-D4 region of the Fbp of S. aureus J2385. Staphylococcus aureus J2385 has been deposited at the National Collection of Industrial and Marine Bacteria Ltd. (NCIMB), Aberdeen, Scotland under number NCIMB 40532 on 18 th December 1992. The D1-D4 region has the amino acid sequence set out in Table 2 below. This new Fbp and D1-D4 polypeptide and antigenically or immunologically equivalent derivatives thereof form a further aspect of this invention. The term 'antigenically equivalent derivative' as used herein encompasses a peptide or its equivalent which will be specifically recognised by certain antibodies which, when raised to peptides according to the present invention, block adhesion of staphylococci to in-dwelling medical devices. The term 'immunologically equivalent derivative' as used herein encompasses a peptide or its equivalent which when used in a suitable formulation to raise antibodies in a vertebrate, die antibodies act to block adhesion of staphylococci to in- dwelling medical devices. In particular derivatives which are slightly longer or slightly shorter than the peptide of the present invention may be used. In addition , peptides in which one or more of the amino acid residues are modified before or after the peptide is synthesised may be used. Such peptides may, for example, be prepared by substitution, addition, or rearrangement of amino acids or by chemical modification thereof. All such substitutions and modifications are generally well known to those skilled in the art of peptide chemistry. The D1-D4 polypeptide may be obtained by expression in Escherichxa coli of the plasmid pBROC520. The preparation of this plasmid and the expression and purification of D 1-D4 polypeptides are described below in die Examples. The DNA encoding this polypeptide is anotiier aspect of this invention. The nucleotide sequence is shown in Table 1 below. Other D1-D4 polypeptides e.g. of Fbp A and FbpB, can be similarly expressed by analogous preparation of appropriate plasmids from chromosomal DNA. The DNA encoding die D1-D4 polypeptide of Fbp A is shown in Table 1 below. The D1-D4 polypeptide, or an antigenically or immunologically equivalent polypeptide or a fusion protein thereof is used as an antigen to immunize a mouse or other animal such as a rat or chicken. The fusion protein may provide stability to the polypeptide. The antigen may be associated, for example by conjugation , witii an immunogenic carrier protein for example bovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH). Alternatively a multiple antigenic peptide comprising multiple copies of die the D1-D4 polypeptide , or an antigenically or immunologically equivalent polypeptide diereof may be sufficiendy antigenic to improve immunogenicity so as to obviate the use of a carrier. Using the procedure of Kohler and Milstein (1975 Nature 256 , 495-497), antibody-containing cells from me immunised mammal are fused with myeloma cells to create hybridoma cells secreting monoclonal antibodies. The hybridomas are screened to select a cell line with high binding affinity and favorable cross reaction widi other staphylococcal species using one or more of the original Fbp, D1-D4 polypeptide and/or die fusion protein. The selected cell line is cultured to obtain the desired Mab. Hybridoma cell lines secreting the monoclonal antibody are another aspect of this invention. Alternatively phage display technology could be utilised to select antibody genes with binding activities towards Fbp or D1-D4 either from repertoires of PCR amplified v-genes of lymphocytes from humans screened for possessing anti-Fbp or from naive libraries (McCafferty, J. et al., (1990), Nature 348, 552-554; Marks, J. et al., (1992) Biotechnology 10, 779-783). The affinity of these antibodies can also be improved by chain shuffling (Clackson, T. et al., (1991) Nature 352, 624-628). Preferably the antibody or derivative thereof is modified to make it less immunogenic in the patient. For example, if the patient is human die antibody may most preferably be 'humanised' ; where the complimentarity determining region(s) of the hybridoma-derived antibody has been transplanted into a human monoclonal antibody , for example as described in Jones, P. et al (1986), Nature 321, 522-525 or Tempest et α/.,(1991) Biotechnology 9, 266-273. The modification need not be restricted to one of 'humanisation' ; other primate sequences (for example Newman, R. et al .1992, Biotechnology, 10, 1455- 1460) may also be used. The antibody should be screened again for high affinity to Fbp, D1-D4 polypeptide and/or fusion protein. As mentioned above, a fragment of die final antibody may be prepared. The humanised monoclonal antibody, or its fragment having binding activity, form a further aspect of this invention. In another broad aspect die invention provides an isolated D1-D4 polypeptide from a Staphylococcus aureus Fbp and its use in die prevention of adhesion of bacteria, in particular gram positive bacteria, to extracellular matrix proteins on in¬ dwelling devices or to extracellular matrix proteins in wounds. The invention further relates to the manufacture of a medicament for such uses. In particular the gram positive bacteria include staphylococci such as S. aureus and coagulase-negative staphylococci, such as S. epidermidis. By isolated D1-D4 polypeptide is meant a polypeptide consisting of the entire Dl, D2, D3 and D4 regions optionally terminating in PIVP and optionally from one to five wall regions (WR) of Staphylococcus aureus Fbp, in sequence. Depending upon the host expression system, the polypeptide may include an N-terrninal methionine residue. The above regions correspond to the following regions of FbpA as described in Signas et al. (1989), op. cit.: D4 G824-P838 WR4 P885-K898 WR5 P899-K912 In a preferred aspect, the polypeptide contains up to three wall regions. Preferred embodiments consist of residues corresponding to residues G709 to T886 and G709 to P838 (optionally where P838→T) of Staphylococcus aureus FbpA. The Fbp is preferably from Staphylococcus aureus J2385 with sequence given in Table 2. This invention also provides isolated nucleic acid molecules encoding the polypeptide, including mRNAs, DNAs and cDNAs. This invention also provides recombinant vectors, such as cloning and expression plasmids useful as reagents in the recombinant production of the polypeptides as well as recombinant prokaryotic and/or eukaryotic host cells comprising the novel nucleic acid sequence. This invention also provides transgenic non-human animals comprising a nucleic acid molecule encoding die polypeptide. A "replicon" is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo: Le., capable of replication under its own control. A "vector" is a replicon, such as a plasmid, phage, or cosmid, to which another DNA segment may be attached so as to bring about the replication of die attached segment A "double-stranded DNA molecule" refers to die polymeric form of deoxyribonucleotides (bases adenine, guanine, diymine, or cytosine) in a double- stranded helix, both relaxed and supercoilecL This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing die structure of particular double-stranded DNA molecules, sequences may be described herein according to d e normal convention of giving only the sequence in the 5' to 3' direction along die nontranscribed strand of DNA (Le., the strand having the sequence homologous to die mRNA). A DNA "coding sequence of or a "nucleotide sequence encoding" a particular protein, is a DNA sequence which is transcribed and translated into a polypeptide when placed under the control of appropriate regulatory sequences. A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining the present invention, die promoter sequence is bound at die 3' terminus by a translation start codon (e.g., ATG) of a coding sequence and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Witiiin the promoter sequence will be found a transcription initiation site (conveniendy defined by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain "TATA" boxes and-"CAT" boxes. Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the - 10 and -35 consensus sequences. DNA "control sequences" refers collectively to promoter sequences, ribosome binding sites, polyadenylation signals, transcription termination sequences, upstream regulatory domains, enhancers, and die like, which collectively provide for die expression (Le., the transcription and translation) of a coding sequence in a host cell. A control sequence "directs the expression" of a coding sequence in a cell when RNA polymerase will bind die promoter sequence and transcribe the coding sequence into mRNA, which is then translated into the polypeptide encoded by die coding sequence. A "host cell" is a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous DNA sequence. A cell has been "transformed" by exogenous DNA when such exogenous DNA has been introduced inside d e cell membrane. Exogenous DNA may or may not be integrated (covalendy linked) into chromosomal DNA making up die genome of die cell. In prokaryotes and yeasts, for example, the exogenous DNA may be maintained on an episomal element, such as a plasmid. Wϊtii respect to eukaryotic cells, a stably transformed or transfected cell is one in which die exogenous DNA has become integrated into the chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by die ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cell containing the exogenous DNA. A "clone" is a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of a primary cell tiiat is capable of stable growth in vitro for many generations. A "heterologous" region of a DNA construct is an identifiable segment of DNA witiiin or attached to anodier DNA molecule that is not found in association witii die otiier molecule in nature. This invention provides an isolated nucleic acid molecule encoding die polypeptide. The isolated nucleic acids particularly the DNAs can be introduced into expression vectors by operatively linking the DNA to the necessary expression control regions (e.g. regulatory regions) required for gene expression. The vectors can be introduced into d e appropriate host cells such as prokaryotic (e.g., bacterial), or eukaryotic (e.g. yeast, insect or mammalian) cells by methods well known in the art (Ausubel et al..supra).The coding sequences for the desired proteins having been prepared or isolated, can be cloned into a suitable vector or replicon. Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice. Examples of recombinant DNA vectors for cloning and host cells which they can transform include d e bacteriophage λ E. £2li), pBR322 (E. £Ωtt), pACYC177 (E- £Ωli), pKT230 (gram-negative bacteria), pGV1106 (gram-negative bacteria), pLAFRl (gram-negative bacteria), pME290 (non-E. £s_H gram-negative bacteria), pHV14 (E- coji and Bacillus subtilis,). pBD9 (Bacillus), pϋόl (Streptomvces). pUC6 (Streptomvces). YIp5 (Saccharomvces). a baculovirus insect cell system, , YCpl9 (Saccharomvces). See. generally. "DNA Cloning": Vols. I & U, Glover et al. ed. IRL Press Oxford (1985) (1987) and; T. Maniatis £i al. ("Molecular Cloning" Cold Spring Harbor Laboratory (1982). The gene can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein as "control" elements), so that die DNA sequence encoding the desired protein is transcribed into RNA in die host cell transformed by a vector containing this expression construction. The coding sequence may or may not contain a signal peptide or leader sequence. The polypeptides of die present invention can be expressed using, for example, the E- coli tac promoter or the protein A gene (spa) promoter and signal sequence. Leader sequences can be removed by the bacterial host in post-translational processing. ≤££. e.g.. U.S. Patent Nos. 4,431,739; 4,425,437; 4,338,397. In addition to control sequences, it may be desirable to add regulatory sequences which allow for regulation of die expression of die protein sequences relative to die growth of die host cell. Regulatory sequences are known to those of skill in the art, and examples include those which cause d e expression of a gene to be turned on or off in response to a chemical or physical stimulus, including die presence of a regulatory compound. Other types of regulatory elements may also be present in the vector, for example, enhancer sequences. An expression vector is constructed so that the particular coding sequence is located in die vector with die appropriate regulatory sequences, the positioning and orientation of die coding sequence with respect to the control sequences being such that die coding sequence is transcribed under the "control" of the control sequences (i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence). Modification of the coding sequences may be desirable to achieve this end. For example, in some cases it may be necessary to modify die sequence so tiiat it may be attached to die control sequences witii die appropriate orientation; i.e., to maintain the reading frame. The control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector, such as the cloning vectors described above. Alternatively, die coding sequence can be cloned direcdy into an expression vector which already contains the control sequences and an appropriate restriction site. In some cases, it may be desirable to add sequences which cause d e secretion of the polypeptide from die host organism, with subsequent cleavage of the secretory signal. A number of prokaryotic expression vectors are known in the art. See, e.g.. U.S. Patent Nos. 4,578,355; 4,440,859; 4,436,815; 4,431,740; 4,431,739; 4,428,941; 4,425,437; 4,418,149; 4,411,994; 4,366,246; 4,342,832; ≤££ aj≤o. U.K. Patent Applications GB 2,121,054; GB 2,008,123; GB 2,007,675; and European Patent Application 103,395. Yeast expression vectors are also known in the art. 2≤£, e.g.. U.S. Patent Nos. 4,446,235; 4,443,539; 4,430,428; ≤££ a]sΩ European Patent Applications 103,409; 100,561; 96,491. pSV2neo (as described in J. Mol. Appl. Genet. 1 :327-341) which uses the S V40 late promoter to drive expression in mammalian cells or pCDNAlneo, a vector derived from pCDNAKMol. Cell Biol. 7:4125-29) which uses the CMV promoter to drive expression. Both tiiese latter two vectors can be employed for transient or stable(using G418 resistance) expression in mammalian cells. Insect cell expression systems, e.g., Drosophila. are also useful, see for example, PCT applications WO 90/06358 and WO 92/06212 as well as EP application EP0290261. Depending on the expression system and host selected, the polypeptide of die present invention may be produced by growing host cells transformed by an expression vector described above under conditions whereby the polypeptide of interest is expressed. The polypeptide is then isolated from die host cells and purified. If the expression system secretes the polypeptide into growth media, die polypeptide can be purified direcdy from the media. If die polypeptide is not secreted, it is isolated from cell lysates or recovered from die cell membrane fraction. The selection of the appropriate growth conditions and recovery methods are within the skill of the art. Another aspect of the invention is a pharmaceutical composition comprising d e above D1-D4 polypeptide or Mab or active fragment and a pharmaceutically acceptable carrier. In therapy or as a prophylactic, the D1-D4 polypeptide or Mab or fragment may be administered to a patient as an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic. Alternatively the composition may be formulated for topical application for example in the form of ointments, creams, lotions, eye ointments, eye drops, ear drops, moudiwash, impregnated dressings and sutures and aerosols, and may contain appropriate conventional additives, including, for example, preservatives, solvents to assist drug penetration, and emollients in ointments and creams. Such topical formulations may also contain compatible conventional carriers, for example cream or ointment bases, and'edianol or oleyl alcohol for lotions. Such carriers may constitute from about 1% to about 98% by weight of the formulation; more -usually they will constitute up to about 80% by weight of the formulation. The composition of the invention may be administered by injection to achieve a systemic effect against relevant bacteria shortly before insertion of an in-dwelling device. Treatment may be continued after surgery during the in-body time of die device. In addition, die composition could also be used to broaden perioperative cover for any surgical technique to prevent staphylococcal wound infections. Many orthopaedic surgeons consider that patients with prosthetic joints should be considered for antibiotic prophylaxis before dental treatment that could produce a bacteraemia. Late deep infection is a serious complication sometimes leading to loss of die prosd etic joint and is accompanied by significant morbidity and mortality. It may therefore be possible to extend die use of die D1-D4 polypeptide or a dierapeutic monoclonal antibody as a replacement for prophylactic antibiotics in this situation. For administration to human patients, it is expected tiiat the daily dosage level of die active agent will be from 0.01 to 10 mg kg, typically around 1 mg/kg. The physician in any event will determine d e actual dosage which will be most suitable for an individual patient and will vary with the age, weight and response of die particular patient. The above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within die scope of this invention. Widi the indicated dose range, no adverse toxicological effects will be observed widi die compounds of die invention which would preclude their administration to suitable patients. In addition to die dierapy described above, the compositions of is invention may be used generally as a wound treatment agent to prevent adhesion of bacteria to matrix proteins, especially fibronectin, exposed in wound tissue and for prophylactic use in dental treatment as an alternative to, or in conjunction with, antibiotic prophylaxis. Alternatively, die composition of die invention may be used to bathe an indwelling device immediately before insertion. The active agent will preferably be present at a concentration of lμg ml to lOmg/ml for badiing of wounds or indwelling devices. The Mabs or fragments described above may also be used as diagnostic reagent to detect the presence of bacteria containing Fbp, of Fbp itself, or die D1-D4 polypeptide of Fbp. Also while the monoclonal antibody aspects of this invention have been described primarily in terms of using the D1-D4 polypeptide of Fbp to generate the antibodies, clearly Fbp itself can also be used as antigen in die initial immunisation programme. The following Examples illustrate the preparation of D1-D4 polypeptides for use as antigens and as anti-adhesive agents. — EXAMPLES Key reagents used in the preparation, isolation and analysis of D1-D4 polypeptides and referred to in the Examples a) Construction of vector pBROC413 The plasmid pT7-7 [Tabor, S (1990), Current Protocols in Molecular Biology , F.A.Ausubel, Brent,R.E. Kingston,D.D. MooreJ.G. Seidman J.A. Smith, and K.Struhl,eds.] pp.16.2.1-16.2.11. Greene Publishing and WUey-lhterscience,New York.] contains DNA corresponding to nucleotides 2065-4362 of pBR322 and like pBR322 can be mobilized by a conjugative plasmid in die presence of a third plasmid ColK. A mobility protein encoded by ColK acts on die nic site at nucleotide 2254 of pBR322 initiating mobilization from this point pT7-7 was digested widi Lspl and BglTL and die protruding 5 * ends filled in widi die Klenow fragment of DNA Polymerasel. The plasmid DNA fragment was purified by agarose gel electrophoresis, the blunt ends ligated together and transformed into E.coli DH1 by electroporation using a Bio-Rad Gene Pulser and following die manufacturers recommended conditions. The resultant plasmid pBROC413 (Fig.1) was identified by restriction enzyme analysis of plasmid DNA. The deletion in pBROC413 from the Lspl site immediately upstream of the φ 10 promoter to the BglK site at nucleotide 434 of pT7-7 deletes die DNA corresponding to nucleotides 2065-2297 of pBR322. The nic site and adjacent sequences are dierefore deleted making pBROC413 non mobilizable. b) Preparation of biotinylated fibronectin probe Fibronectin (FN) was purified from a human plasma fraction (a gift from Dr. D. Pepper, Scottish Blood Transfusion Service) on Gelatin Sepharose, essentially as described by Miekka et al (1982) Thromb. Res. 27, 1-14. 11.2mg FN (2.0ml) in 0.05M Tris/O.IM NaCl pH7.5 was made l.Omg/ml by addition of 9.2ml 0.05M Na2B4 7 ρH8.6 buffer. 80μl of N-hydroxysuccinimidobiotin (Amersham UK) from a 5.0mg/ml stock in dry dimediylformamide was added and the mixture incubated at room temperature for lh with constant agitation. The reaction was terminated by buffer exchange into Dulbecco's 'A' phosphate-buffered saline containing 0.1% ^ ) bovine serum albumin (BSA) using five Sephadex G25 columns (PD10, Pharmacia). c) Synthesis of Fibronectin Sepharose CL4B 100 mg of human fibronectin, purified as described above , was coupled to 7 g of Cyanogen bromide-activated Sepharose CL4B (Pharmacia) at room temperature according to die manufacturers instructions, to yield 25 ml of gel matrix. Before use the FN-Sepharose was washed wid all buffers used in d e subsequent purification. Numbering of amino acid residues in the Examples In the following examples the numbering of amino acid residues corresponds to die residues of FbpA according to Signas et al., (1989) op. cit. Residues 709-838 of FbpA correspond to residues 1-130 of die sequence of S. aureus J2385 given in Table 2 and residues 709-886 correspond to residues 1-174. Example 1 Isolation of DNA from Staphylococcus aureus J2385 coding for the fibronectin binding domains of Fibronectin Binding Protein. S. aureus J2385 is strain B described in Cookson et al. [1987] THE LANCET of August 15th. page 387. It is a clinical strain derived from a skin lesion. Chromosomal DNA was prepared by treating cells collected from an overnight shake flask culture widi lysostaphin to lyse them and phenol/chloroform to remove cell protein. From this unpurified DNA preparation the fragment of DNA encoding the fibronectin binding domains of Fibronectin Binding Protein was obtained by a PCR amplification reaction. The oligonucleotide primers used in the PCR reaction were:- FLB 1 S'-GGGAATTCATATGGGCCAAAATAGCGGTAACCAGTC-S' FIB 2 5 , -GCGGATCCTTACGTTGGTGGCACGATTGGAGGTG-3 , PCR amplification was carried out using 5. aureus J2385 chromosomal DNA (lOng.) FIB 1 (1 micromolar), FIB 2 (1 micromolar), Tris-HCl pH8.3 (10 mM), KCl (50mM), MgCl2 (1.5 mM), gelatin (0.001%), Na dGTP (200 micromolar) Na dATP (200 micromolar), Na dTTP (200 micromolar), Na dCTP (200 micromolar) and Taq DNA Polymerase (2.5 units) in a final volume of 100 microlitres made up widi distilled water. The aqueous solution was overlaid widi 80 microlitres of liquid paraffin and subjected to 30 cycles of 94 °C (lmin.), 60°C (1 min.) and 72°C (2 min.) to enable die amplification to occur. When 10 microlitres of the aqueous reaction were examined after amplification on 1.5% agarose gel electrophoresis in the presence of ethidium bromide (0.5 micrograms/mL) it was observed by comparison to a sample of DNA fragments of known size that a single species of DNA fragment of approximately 500 basepairs was obtained. Example 2 Obtaining the sequence of the DNA fragment obtained by PCR amplification of S. aureus J2385 chromosomal DNA using primers FIB 1 and FIB 2. The size of the PCR fragment obtained (approx. 500 bp.) using conditions described in Example 1 was unanticipated as die primers FIB 1 and FIB 2 were designed to be homologous to sites in Fibronectin Binding Protein gene (as described by Signas C, et al. [1989] P.N.A.S. USA vol 86,699-703) of 5. aureus which were reported to exist approximately 400 bp. apart In order to aud enticate the nature of the DNA fragment it was cloned into ρUC19 and sequenced. The DNA from one PCR reaction was incubated with Eco RI and Bam HI restriction enzymes and cloned into similarly treated pUC19 [Yanisch -Perron, C. et al. (1985) Gene, 33, 103] and sequenced to create pBROC 519a. Because the PCR process occasionally introduces deletions and base substitutions into amplified DNA, similar clones of J2385 DNA were retained to cosequence along with pBROC 519a for confirmation of results. pBROC 519a was sequenced in bodi strands using die SEQUENASE II kit obtained from United States Biochemical. The sequence obtained revealed die cloned fragment encoded 524bp of 5. aureus J2385 DNA. When die sequence was compared widi tiiat published by Signas et al. (loc. cit.) it was apparent that it was extensively homologous to region 2350-2885 of that S. aureus gene (using the Signas et al. numbering) but possessed significant differences (see Table 1). In particular, die derived amino acid sequence displayed amino acid differences to die published sequence at residues 752 (ASN→ASP), 803 (SER→ASN), 821 (LYS→GLN), 825 (GLN→HIS) and a four amino acid deletion (838→841), see Table 2. These differences were confirmed by sequencing independent clones of PCR amplified DNA. When die derived amino acid sequence from 5. aureus J2385 DNA was compared to die corresponding regions in S. aureus Fibronectin binding protein type A (Signas et al. [1989]) and 5. aureus type B protein (Jonsson et al. [1991] EUR. J. BIOCHEM. vol 202 pages 1041-1048) it was clear tiiat die fibronectin binding domains of die S. aureus J2385 protein was very similar to the respective domains of both the type A and type B proteins but was identical to neitiier (Table 2). This suggested that the 5. aureus J2385 protein should be denoted type C. Example 3 Preparation of pBROC 520 in £. coli BL21 (DE3) In order to express die protein fragment encoded by d e cloned S. aureus J2385 DNA die T7 polymerase/promoter expression system as described by Tabor,S. was used (see Current Protocols in Molecular Biology [F.A.Ausubel, R.Brent, R.E.Kingston, D.D.Moore, J.G.Seidman, J.A.Smith and K.Struhl,eds.] pp.16.2.1 - 16.2.11. Greene Publishing and Wiley-Interscience, New York). The host was E.coli BL21(DE3) which inducibly expresses the T7 polymerase gene (see Studier F.W. and Moffat B.A. [1986] J.Mol.Biol. vol.189,113-130). The 0.5 kb BamHl/Ndel fragment of Sxuireus J2385 derived DNA was isolated from pBROC 519a (3 microgrammes) by restriction enzyme digestion and electrophoresis on low melting point agarose gel. This material (100 nanogrammes) was used in a ligation reaction widi BamHl/Ndel digested pBROC 413 DNA (500 nanogrammes). The ligated DNA was electrotransformed into E.coli Delta M15 (see Sambrook, J., Fritsch, E.F. and Maniatis, T. editors [1989] Molecular Cloning, A Laboratory Manual (second edition) page 2.57 for details of die lacZ Delta M15 mutation) and transformants selected on LB agar containing ampicillin (50 microgrammes/ml.). Plasmid DNA preparations were made from five ampicillin resistant colonies and were all shown by restriction enzyme site mapping to be pBROC 413 carrying the S. aureus derived DNA fragment. One of die plasmid preparations (designated pBROC 520) was used to transform Exoli BL21(DE3) to give the desired expression construct host combination. pBROC 520 encodes die peptide given in SEQ ID NO 6, hereinafter referred to as Dl-D4(709-886). Example 4 Expression, isolation and purification of D1-D4 (709-886) polypeptide of J2385 expressed from pBROC 520 in E. coU BL 21 (DE3) a) Expression Single colonies of E. coli BL 21 (DE3) harbouring either pBROC 413 (non- coding) plasmid or pBROC 520 (encoding D1-D4 (709-886)) were inoculated into 30 ml capped containers (universals) containing 10 ml of NZCYM medium (1% (w/v) Bactotryptone, 0.5% (w/v) Bacto yeast extract, 0.5% (w/v) NaCl, 0.1% (w/v) casamino acids and 0.2% (w/v) MgSO.}- 7 H2O pH 7.0) and 75 μg/ml ampicillin. The cultures were incubated at 37°C , 230 rpm overnight The overnight cultures were used to inoculate 250 ml NZCYM medium containing 150 μg/ml ampicillin. The cultures were incubated at 37°C, 230 rpm until A 00 reached 0.5 absorbance units. The cultures were then induced widi lmM IPTG (Isopropylthio-β-D- galactoside) and incubated under the same conditions for a further 4 hours. 1 ml samples were removed pre-induction and 1,23 and 4 hours post-induction. Each sample was spun in an eppendorf centrifuge for 1 minute after which the supernatant was removed. The pellets were then resuspended in lOOμl of reducing buffer (50mM Tris. HC1 pH6.8, lOOmM dithiothreitol (DTT), 0.1% (w/v) bromophenol blue, 2% (w/v) SDS, 10% (v/v) glycerol) or non-reducing buffer (DTT is omitted). The samples were heated for 3 mins at 90°C before being stored at -40°C b) Detection of expressed product E. coli resuspended pellets containing either pBROC 413 (non-coding plasmid) or pβROC 520 (encoding D 1-D4 (709-886)) were separated on sodium dodecyl sulphate containing 4-20% polyacrylamide gels (Novex, British Biotechnology Ltd.), essentially as described by d e manufacturers. Separated proteins were transferred to Immobilon (Millipore (UK) Ltd.) using Sartoblot II blotting apparatus according to the manufacturer's instructions. Unreacted sites on the blot were blocked by incubation in lOmM Na^PO O.15M NaCl/0.02%( v) Ficoll/0.02%( /v) polyvinylpyrrolidone/0.1%( w v) bovine serum albumin (BSA) pH7.4 for lh at room temperature with constant agitation. The blot was probed widi biotinylated fibronectin at 200 μg ml in 0.02M NaH 2 PO4/0.3M NaCl/0.5%( v) Tween 80/1.0%( /v) BSA pH7.4 for 4h at room temperature with constant agitation. Bands were visualised using a Streptavidin Gold/silver stain system (Amersham UK) according to die manufacturer's instructions. Putative D1-D4 (709-886) polypeptide was identified as a new band in die pBROC 520 lane. c) Isolation of solubilised D1-D4 (709-886) Frozen cell pellet of E. coli BL21 DE3 (pBROC 520) ) (from a 300 ml culture) prepared essentially as described in a) above and using an induction period of 3h was allowed to thaw at 4 °C for 2h and was then resuspended in 50 mM Tris/50 mM NaCl/1 mM EDTA/0.1 mM phenyl methyl sulphonyl fluoride (PMSF) pH 8.0 (30 ml). The suspension was transferred to a 100ml glass beaker and sonicated (Heat Systems - Ultrasonics W380; 70 Watts, 50 x 50 % pulse, pulse time = 5 sec). The sonicate was immediately centrifuged (6000g / 4°C/ 10 min) and die pellet discarded. The supernatant containing die solubilised D1-D4 (709-886), was adjusted to pH 7.4 and retained at -40°C. d) Purification of D1-D4 (709-886) product (i) D1-D4 (709-886) supernatant prepared as described above, was applied to a FN-Sepharose column (1.6 x 13.2 cm) equilibrated in Dulbecco's 'A' phosphate- buffered saline (PBS)/0.4 M NaCl/0.1 mM PMSF. D1-D4 (709-886) was eluted from the column with PBS/2M GuanidincHCI and tiien concentrated by stirred cell ultrafiltration, using a M r 10,000 cut-off membrane (Amicon), to a 4.0 ml retentate. The D1-D4 (709-886) retentate was formulated into product by buffer exchange into PBS using two Sephadex G25 columns (PD10, Pharamacia). 1.5 mg >90% pure, determined by RP-HPLC and SDS PAGE, D1-D4 (709-886) product was obtained; material was confirmed as D1-D4 (709-886) by N-terminal sequencing and * by Western blotting (probed with biotinylated fibronectin). The molecular weight of tfae isolated, purified polypeptide , determined by electrospray mass spectrophotometry was 19,970. The theoretical molecular weight is 19,969. Molecular weight analysis according to SDS PAGE indicated that the D1-D4 (709-886) polypeptide had a mobility corresponding to a protein of approximately 35000 (non-reduced) or approximately 40000 (reduced) ; die protein markers used for this were the Low Molecular Weight Kit (Pharmacia). (ii) An alternative purification metiiod was also developed. D1-D4 (709-886) supernatant (from a 5L culture), prepared essentially as described in Example 4c, was diluted 1:1 in 0.1M NaH2PO4 pH 7.6 (final pH adjusted to 7.6) and applied to a Q Sepharose (Pharmacia) column (i.d.,7.8cm; h,5cm) equilibrated in 0.1M NaH 2 PO 4 pH 7.6. D1-D4 (709-886) adsorbed to the column and was eluted using 0.1M NaH2PO4 / 0.5 M NaCl pH 7.6. It was tiien concentrated by stirred cell ultrafiltration, using a M r 10,000 cut-off membrane (Amicon), to a 30ml retentate. At this stage D1-D4 (709-886) was approx.50% pure. TheDl-D4 (709-886) retentate was buffer exchanged into 50mM formic acid using a Sephadex G25 (Pharmacia) column (i.d.,2.6cm; h,21cm) to yield a 40ml product The D1-D4 (709-886) product was further purified by four repeat runs on reverse-phase HPLC Thus, 10ml D1-D4 (709-886) was applied to an Aquapore C4 column (Applied Biosystems) (i.d.,lcm; h,10cm) equilibrated in 0.1% trifluoroacetic acid (TFA). Dl- D4 (709-886) was eluted from the column using a 0 to 100% linear gradient of 0.085% TFA / 70% acetonitrile, over 4-5 column volumes. The appropriate D1-D4 (709-886)-containing fractions from the four repeat runs were pooled and were concentrated by ultrafiltration (as above) to a 30ml retentate. The D1-D4 (709-886) retentate was formulated into final product by buffer exchange into 50mM formic acid (as above) followed by lyophilisation. 50mg >95% pure, determined by analytical reverse-phase HPLC and SDS PAGE, D1-D4 (709-886) product was obtained; material was confirmed as D1-D4 (709-886) by N-terminal sequencing and by Western blotting (probed with biotinylated fibronectin). The solubility of the D1-D4 (709-886) lyophilised product was 35-40mg/ml when reconstituted in H2O or 50mM formic acid. (iii) A second alternative purification metiiod was developed to replace die C4 reverse-phase step. D1-D4 (709-886) (48ml) that had been eluted from a Q Sepharose column as described in (ii) was mixed wid 4M (NH 4 )2SO 4 (16ml) and applied to a Toyopearl Butyl column (TosoHaas) (i.d., 1.6cm; h, 15cm) equilibrated in 1.0M (NH4) 2 SO4 / 0.1M NaH PO pH7.0 (Buffer A). The column was then washed with approx. 3 bed volumes of Buffer A. The D 1-D4 (709-886), which adsorbed to the matrix, was eluted from the column using a 30% to 100% linear gradient of 0.1M NaH 2 PO pH7.0 in Buffer A over 3 column volumes. The appropriate D1-D4 (709-886)- containing fractions were identified by SDS PAGE, were pooled and were then concentrated by ultrafiltration, using a M r l 0,000 cut-off membrane (Amicon), to a 20ml retentate. The D1-D4 (709-886) retentate was formulated into final product by buffer exchange into 50mM formic acid using a Sephadex G25 column (Pharmacia) (i.d., 2.6cm; h, 21cm) followed by lyophilisation. 50mg >98% pure, determined by reverse-phase HPLC and SDS PAGE, Dl- D4 (709-886) product was obtained. Example 5 Bioreactor fermentation of D1-D4 (709-886) polypeptide Single colonies of E.coli BL21 (DE3):pBROC520 were recovered from LB agar medium containing 50μg/ml ampicillin and used to inoculate 2x100 ml of seed medium (NCYZM) containing ampicillin at 75μg/ml. The primary and secondary seed stage fermentations were carried out in 500ml shake flasks batched with 100ml aliquots of NCYZM medium. The primary and secondary seed fermentation conditions were as follows: 37°C, 230 rpm on an orbital shaker with a 50mm throw. The primary seed incubation time was 9 hours. The primary seed culture was used to inoculate (0.1% v/v) 6x100 ml aliquots of secondary seed stage medium (NCYZM). The secondary seed was incubated for 14.5 hours. Two 15 litre Biolafitte fermenters were each batched with 10 litres of NCYZM medium and 0.01 % (v/v) Dow Coming DC 1510 antifoam. The vessels plus media were sterilised using steam to 121°C for 45 minutes. Ampicillin sterilised by microfiltration (0.2μm) was added aseptically to die vessel media to a final concentration of 150μg/ml. The fermenters were inoculated at a level of 2.5% (v/v) from pooled secondary seed culture. The final stage incubation conditions were 37°C, agitator 300 rpm, airflow 5 1/min (0.5wm). The final stage fermentations were sampled aseptically pre-inoculation, at 0 hours and thence approximately hourly. The samples were monitored for increases in optical density (550nm). When the OD 550 was >= 1.0, IPTG was added to give a final concentration of lmM. The fermentations were incubated for approximately a further 3 hours. The cells were recovered by batch centrifugation using 7000g for 35 minute, or continuous centrifugation at 15,000g. The total cell yield was 73.5 grammes. Cells were washed once with a total of 1.0 litre of Oxoid phosphate buffered saline (Dulbecco 'A') pH 7.2. The centrifuged washed cells were then held frozen at -20°C to await further processing. pH adjusted to 7.0 widi 5M NaOH, tiien final volume to 1000ml. Example 6 Construction of pBROC 533, a plasmid expressing a polypeptide predominantly consisting of the D1-D4 domains of S.aureus J2385 Fibronectin Binding Protein. The following oligomeric polynucleotide (A) and its complement were synthesised on a Pharmacia LKB Gene Assembler Plus DNA synthesiseπ- CGGAATTCGT CAACAAACGA TTGAAGAAGA TACAACGACG TAAGATCTGG ATCCGCATGC GAATTCCG Samples of the two oligonucleotide preparations (0.14 microgrammes of each) were mixed, heated to 94 degrees C for 5 minutes and tiien allowed to anneal at 37 degrees C for 10 minutes. The double stranded DNA was digested with EcoRI enzyme and subsequendy cloned into d e EcoRI site of pUC19 (2 microgrammes of EcoRI digested vector/ligation) to give pBROC 528 in E. coli Delta M15. The BamHI digested kanamycin resistance marker of pUC4K (obtained from Pharmacia, code no. 27-4958-01) was tiien cloned into die unique Bgiπ site of the resulting plasmid construct pBROC 528 to give pBROC 529. pBROC 529 plasmid DNA (5microgrammes) grown in E. coli Delta M15 was double digested widi HincII/SphI and the approximately 1.4kb DNA fragment encoding die kanamycin resistance gene and tiiat moiety of Fibronectin Binding Protein encoded by die oligonucleotide originally syntiiesised was isolated using low melting point agarose/agarase. This DNA was used in a ligation reaction widi partially digested HincIL completely digested SphI plasmid DNA (1 microgramme) of pBROC 519a (see Example 2). The ligation products were electrotransformed into E. coli Delta Ml , selecting for kanamycin resistance. In this way, it proved possible to isolate pBROC 530, a plasmid which as ascertained by sequencing across the Hincll sites of the staphylococcal DNA and by restriction mapping, carried a DNA fragment encoding the D1-D2-D3-D4 regions (residues 1-129 in Table 2) of die Fibronectin Binding Protein of S. aureus J2385. The DNA fragment additionally coded for a tiireonine residue at die carboxy terminal of the polypeptide. Next, the staphylococcal DNA of pBROC 530 was removed from the plasmid vector by Ndel/BamHI digestion of a plasmid DNA preparation and cloned into similarly digested pBROC413 (see Key reagents a) to give pBROC 531. pBROC 531 was grown in a transformed strain of E. coli Delta M 15 and then digested widi Sail to remove the kanamycin resistance gene and subsequendy religated to create pBROC 533. This step was carried out to prevent unnecessary overexpression of the kanamycin resistance gene from the T7 promoter in pBROC 531 as it was considered tiiat this would be detrimental to maximal expression of the staphylococcal DNA. pBROC 533 was transformed into E.coli BL21(DE3) to furnish E oli BL21(DE3), pBROC533. A variant of plasmid pBROC 531 may alternatively be constructed using die following oligomeric polynucleotide (B) and its complement: - CGGAATTCGT CAACAAACGA TTGAAGAAGA TACAACGCCG TAAGATCTGG ATCCGCATGC GAATTCCG This gives the same polypeptide as above with die terminal threonine replaced by proline. Example 7 Expression, isolation and purification of D1-D4 (709-838(P838T)) polypeptide expressed from pBROC 533 in E.coli BL21(DE3) a) Expression This was carried out using the methods described in Example 4a), except that plasmid pBROC 533 from Example 6 was substituted for pBROC520. b) Detection of expressed product These were carried out using die methods described in Example 4b). D1-D4 (709-838(P838T)) was identified as a new band in the pBROC 533 lane. c) Isolation This was carried out essentially as described in Example 4c), except that cell pellets of E oli BL21 (DE3) (pBROC 533) were used, the starting volume of culture was 1.2 litres and the cell pellet was resuspended in 32ml buffer. The final supernatant volume was 40 ml. d) Purification D1-D4 (709-838(P838T)) supernatant, prepared as described above, was diluted 1:1 with O.IM NaH 2 PO 4 pH7.6, the pH adjusted to 7.6 using HC1 and applied to a Q Sepharose (Pharmacia) column (i.d., 4.1cm; h, 4.7cm) equilibrated in O.IM NaH2PO4 pH7.6. The polypeptide adsorbed to die column and was eluted using O.IM NaH 2 PO 4 / 0.5M NaCl pH7.6. Polypeptide solution (50ml) that had eluted from the Q Sepharose was mixed widi 4M (NH4)2SO4 (16ml) and applied to a Toyopearl Butyl column (TosoHaas) (i.d., 1.6cm; h, 15cm) equilibrated in 1.0M (NH4) 2 SO4 / 0. IM NaH 2 PO 4 pH7.0 (Buffer A). The column was then washed with approx. 3 bed volumes of Buffer A. The polypeptide, which adsorbed to die matrix, was eluted from die column using a 30% to 100% linear gradient of 0.1M pH7.0 in Buffer A over 3 column volumes. The appropriate polypeptide-containing fractions were identified by SDS PAGE, were pooled and were tiien concentrated by ultrafiltration, using a M r 10,000 cut-off membrane (Amicon), to a 20ml retentate. The retentate was formulated into final product by buffer exchange into 50mM formic acid using a Sephadex G25 column (Pharmacia) (i.d., 2.6cm; h, 21cm) followed by lyophilisation. An aliquot of the lyophilisate was solubilised; d e material showed a single major band of approximately M j =22000 on SDS PAGE under non-reducing conditions. BIOLOGICAL EVALUATION A. Effect of D 1-D4 (709-886) on adhesion of staphylococci to fibronectin- coated polymethylmethacrylate (PMMA) coverslips in vitro. (i) Bacterial strains Staphylococcus aureus 8325-4 (standard laboratory strain) Staphylococcus aureus J-2385 Staphylococcus epidermidis (SE 902 and SE 903, two clinical isolates from foreign body infection) (ii) Adhesion assay The in vitro adhesion assay described by Vaudaux et al ., Infection and Immunity 45:768-774, 1984, was used to measure staphylococcal adhesion to fibronectin-coated surfaces and to test die anti-adhesive properties of the D1-D4 (709- 886) polypeptide. (tii) Preparation of bacteria Staphylococcal strains were grown overnight at 37°C in Mueller-Hinton Broth (MHB, Difco Laboratories). A total of 2 x 10 7 cfu from the overnight culture was incubated widi lOOμCi [metAy/- 3 H]-thymidine (Amersham) in 1ml of MHB and grown for 3 hours at 37°C up to l x l0 8 - 2 x l0 8 cfu ml. After removal of the unbound radioactivity by two washes (3,000 x g, 10 minutes) the labelled strain was suspended in 1ml of 0.15M NaCl. (iv) Adsorption of fibronectin to PMMA coverslips All PMMA coverslips (0.75cm x 0.75cm) were immersed in 95% etiianol for 10 minutes at 37°C, drained and heated at 120°C for 30 minutes. The coverslips were then preincubated widi gelatin (lmg/ml, Difco Laboratories) for 60 minutes at room temperature and rinsed with PBS (without Ca 2+ -Mg 2+ , Gibco Laboratories). Coverslips were immersed in solutions containing purified human fibronectin at two concentrations, 0.5μg/ml for S. aureus strains and 4μg/ml for S. epidermidis strains, for 60 minutes at 37°C. Coverslips were transferred to fresh tubes containing PBS and washed. (v) Adhesion assay D1-D4 (709-886) polypeptide was diluted to the required concentration (ranging from lOOμg/ml to lOpg/ml) in PBS supplemented with cations (Gibco Laboratories) and albumin (20%, Sigma) (PBS ++ /ΗSA). Washed radiolabelled culture (40μl) was added to 960μl of the diluted D1-D4 (709-886) polypeptide in plastic tubes. Fibronectin-coated coverslips were added to the bacterial suspensions and incubated at 37°C for 60 minutes with shaking (lOOcycles/minute) Fluids were then drained away, coverslips transferred to fresh tubes, washed twice with saline (1ml) at room temperature for 1x5 and 1x30 minutes. Fluids were drained away and coverslips transferred to scintillation vials supplemented with 5ml scintillation fluid and counted. (vi) Evaluation of cpm and cfu for each strain Portions of the 3 hour radiolabelled culture (lOμl) were counted in scintillation vials. lOOμl Aliquots were diluted 1 in 10,000 and 20μl plated onto Mueller-Hinton agar and viable counted. In addition, the cpm content of the lOOμl of the diluted strain used in die adhesion assay was also checked to enable the numbers of cfu's adhering to fibronectin-coated PMMA coverslips to be calculated. (vii) Results * Coverslips coated with 0.5 μg/ml of fibronectin ** Coverslips coated with 4.0 μg/ml of fibronectin S. aureus 8325-4 is a standard strain which has been widely studied for its ability to adhere to a variety of materials coated widi human fibronectin. The adhesion of mis strain to fibronectin-coated surfaces is promoted when die surface is coated widi increasing concentrations of fibronectin. Therefore, S. aureus 8325-4 was selected as being a suitable strain to test the effects of agents that might block the interaction between staphylococci and fibronectin-coated surfaces. D1-D4 (709-886) is a potent inhibitor of S. aureus 8325-4 adhesion to fibronectin-coated coverslips. Up to 95% adhesion inhibition could still be detected when die concentration of D1-D4 (709-886) was reduced to lOng/ml. Purified human IgG (Pierce) was used as a control, since this has previously been shown to inhibit 5. aureus adhesion to fibronectin-coated surfaces using this assay (Vaudaux et al. J. Invest Surg, 2: 397-408 1989), and gave 70% inhibition at 1 mg/mL In addition, die adhesion of S. aureus J-2385 to fibronectin-coated coverslips is also promoted when ' increasing concentrations of fibronectin are used to coat the PMMA coverslips. However, the gelatin pretreatment of coverslips, described in die standard protocol, was omitted since it resulted in some non-specific binding which interfered widi d e adhesion assay. Demonstrating die adhesion of this strain to immobilised fibronectin is significant since the recombinant D1-D4 (709-886) polypeptide used in tins study was derived from S. aureus J-2385. D1-D4 (709-886) polypeptide is capable of inhibiting adhesion of 5. aureus J-2385 to fibronectin- coated surfaces. The adhesion of 5. epidernάάis was also promoted when die concentration of fibronectin used to coat the coverslip was increased. It was noted tiiat higher concentrations of fibronectin (4μg/ml) were required to allow significant numbers of organisms to adhere when compared with S. aureus 8325-4 (0.5μg/ml). The D 1-D4 (709-886) polypeptide also inhibited adhesion of 5. epidermidis to fibronectin (95% adhesion at 1 μg/ml) suggesting tiiat D1-D4 (709-886) could have anti-adhesive activity as a therapeutic agent against coagulase-negative staphylococci. B. Inhibition of 5. aureus adhesion to dermal substrates (i) Introduction The dermal tissues used in this study was obtained as a kit (Skin 2 ) from Advanced Tissue Sciences (La Jolla, California, USA). Full details on growing the artificial dermis are supplied widi die kit The artificial dermis was formed by seeding human neonatal fibroblasts onto an inert medical grade nylon mesh. The fibroblasts adhere to die mesh and proliferate within the mesh interstices. During tiiis process they secrete naturally occurring growth factors and deposit extracellular matrix proteins such as fibronectin and collagen thus developing into a human dermis. This model has been successfully used to represent die dermal tissues exposed in wounding. (u) In vitro dermal adhesion assay with S. aureus 8325-4 The procedure was essentially identical to that described above in A. except diat samples of artificial dermis replaced the FN-coated coverslips. S. aureus 8325-4 was grown overnight in Mueller-Hinton Broth (MHB, Difco). A total of 2 x 10 7 cfu from the overnight culture was incubated with lOOμCi [ netΛy/- 3 H]-thymidine in 1ml MHB and grown for 3 hours at 37°C up to 1 x 10 8 to 2 x 10 8 cfu ml. After removal of the free radioactivity by two centrifugations (3,000 x g, 10 minutes) the labelled strain was suspended in 0.5 % HSA (Sigma) to prevent non-specific binding to cellular/matrix proteins. The dermal substrates were washed widi serum-free media and PBS prior to die addition of the radiolabelled bacteria. (iii) Effect ofDl-D4 (709-886) on adhesion to dermal substrates D1-D4 (709-886) polypeptide was diluted to the required concentration in PBS supplemented with cations (Gibco Laboratories) and human serum albumin (20%, Sigma) (PBS * " " - HSA). Washed bacteria (40μl) were added to diluted samples of D1-D4 (709-886) (960μl). The dermal tissues were then added to die bacterial suspensions containing D1-D4 (709-886) and incubated at 37°C with shaking (100 cycles/minute). Fluids were drained away, dermal tissues were transferred to fresh tubes, washed with saline (1ml) at room temperature for 5 and 30 minutes respectively. Fluids were drained away and dermal tissues transferred to scintillation vials supplemented wid 5ml scintillation fluid and counted. (iv) Results D1-D4 (709-886) has a concentration-dependent inhibitory effect on the adhesion of S. aureus 8325-4 to dermal tissues. Significant adhesion inhibition was achieved (>50%) at concentrations of D1-D4 (709-886) down to lμg/ml. These results show a good correlation to tiiose obtained widi die fibronectin-coated PMMA coverslip adhesion assay. In addition, die results confirm the importance of die interaction between S. aureus and fibronectin present in dermal tissues. As a consequence of this observation D1-D4 (709-886) polypeptide may also be useful prophylactically as a topical application in the prevention of wound infections during surgery. C. Inhibition of adhesion of staphylococci to nylon cannulae during subcutaneous implantation in the rat. (i) In vivo model of experimental foreign body infection Male CD rats (Charles River (U.K.) Limited) weighing 230-250g were used in this study. Three or four flexible vinyl cannulae (1.5mm x 2cm) were subcutaneously implanted in each rat under sterile conditions. Animals were anaestiietised with Hypnorm (0.05ml) i.m. followed by 0.05 ml i.m. valium. A 1 cm incision was made in centre of back to side of spine and a subcutaneous area was opened by blunt dissection. The cannulae were implanted and die incision was closed wid clips. The wound was sprayed with Xylocaine prior to recovery from anaestiiesia. The clips were removed after 6 days. The implant site was infected by dispensing bacterial culture (see below) into the wound during insertion of the cannulae. (ii) Preparation of bacteria for use in infection model Bacteria were grown overnight in MHB (Difco). Overnight culture (20μl) was used to inoculate fresh MHB (1ml) and die culture was grown at 37°C for 3h or 16h (exponential phase culture). The cells were washed twice(9,000 x g, 10 minutes) arid resuspended in 0.85% PBS (1ml) to give 8.3 log 10 cfu/ml. The bacterial suspensions were diluted 1 in 10 to give approximately 7 log 10 cfu/ml and 0.1ml of this suspension was used to infect the rats. Therefore, each wound was infected widi approximately 6 log 10 cfu. To test the effect of D 1 -D4 (709-886) on adhesion in vivo, the D 1 -D4 (709- 886) polypeptide was added to die bacterial suspension to give a final concentration of 500μg/ml prior to addition to the wound site. Thus, each wound site was dosed with 50μg Dl-D4 (709-886). (Hi) Evaluating bacterial adhesion/colonisation in vivo in the presence ofDl-D4 (709-886) polypeptide At times e.g 1, 6, 24, 72, 168, 192 and 240h post infection, one rat from each group was sacrificed using an i.p. injection of 0.5ml Expiral. All cannulae were removed, rinsed in sterile PBS and each placed in 1ml of fresh PBS (phosphate- buffered saline, 0.9%) and sonicated for 2 minutes in a sonicating water bath to remove adherent bacteria. Samples were plated out on to Mueller-Hinton agar to count the numbers of adherent, viable bacteria. (iv) Results D1-D4 (709-886) polypeptide inhibits adhesion of 5. aureus 8325-4 to cannulae during subcutaneous implantation in the rat Of the cannulae removed after six hours implantation, tiiree out of four were completely free of adherent organisms, and the number of organisms adherent to the fourth cannula was considerably less than the controls where no D1-D4 (709-886) was added. Although some adherent bacteria were detected on cannulae after 8 days of implantation the numbers of organisms associated widi D1-D4 (709-886) treated cannulae (3.0±1.31ogιo cfu) was much lower than the untreated controls (5.4±1.41ogιø cfu). Considering the numbers of organisms used to infect the wound was 10 6 this should exceed the numbers of contaminating organisms present in the operating theatre during surgery. D 1 -D4 (709-886) inhibited adhesion of S. aureus 120 (a virulent clinical isolate) for at least 10 days post infection. Cannulae were removed at 6, 24, 72, 168 and 240h post infection and bacterial counts of between 4 and 6 logjQ cfu were detectable for the duration of the study in the untreated cannulae. However, little or no bacterial numbers were detectable in the D1-D4 (709-886) treated cannulae at any time. Similar results were seen with two S. epidermidis strains. Despite an apparent delay in adherence of these strains, high numbers, between 5 and 6 log^o cfu were detectable at 10 days post infection on untreated cannulae. No bacteria were . detectable on die D1-D4 (709-886) treated cannulae at this time. Therefore, D1-D4 (709-886) is potentially effective in blocking staphylococcal adhesion to indwelling medical devices when used prophylactically as a topical agent. D. Effect of D1-D4 (709-838(P838T)) on adhesion of staphylococci (i) In vitro adhesion assay Assay conditions were identical to those described for die evaluation of Dl- D4 (709-886) polypeptide in A. (ii) Results The D1-D4 (709-838(P838T)) polypeptide was capable of inhibiting the adhesion of bodi S. aureus 8325-4 and 5. epidermidis SE 902 to fibronectin-coated coverslips. The adhesion inhibition achieved with D1-D4 (709-838(P838T)) was equivalent to tiiat observed with D1-D4 (709-886). In tiiis study S. aureus 8325-4 adhesion was inhibited by 95% widi lOOng/ml polypeptide and S. epidermidis SE 902 adhesion inhibited by 85% at lOOμg/ml polypeptide. (iti) In vivo adhesion assay Assay conditions were identical to diose described for the evaluation of Dl- D4 (709-886) polypeptide in C. (iv) Results The D1-D4 (709-838(P838T)) polypeptide was capable of inhibiting the adhesion of S. aureus 120 and S. epidermidis SE902 to vinyl cannulae during suncutaneous implantation in die rat. No adherent organisms were detected on cannulae treated with D1-D4 (709-838(P838T)) up to the 10 day time period studied in contrast to untreated controls where 4.5 logio cfu were detected for S. aureus 120 and 6 log jo cru f° r S. epidermidis SE 902. Table 1 A Comparison of die DNA Encoding die Fibronectin Binding Domains of S. aureus Fibronectin Binding Protein type A and of die S. aureus J2385 DNA sequence. 1 GGCC AA AATAGCGGTAACCAGTCATTCGAGGAAGACACAGAAGAAGATAA SO ~ J2385 I I I ! I I I I I I 1 I I I I I M I I M I I 1 I I I I I 1 I I I I I I II I I 1 I I I U + l I 2350 GGCCAAAATAGCGGTAACCAGTCΛTTCGΛGGΛAGΛCΛCΛGΛΛGAAGACΛA 2399 Type Λ 51 ACCTAAATATGAACAAGGTGGCAΛTΛTCGTΛGΛTATCGΛTTTCGATΛGTG 100 J2385 1 I I I I I I I I I I I I I I I I I I I I 11 I I II I I I I I I I II I I I I 1 l + l I 1 II I I 2400 ACCTAAATATGAACAAGGTGGCAATATCGTAGATATCGΛTTTTGATAGTG 2449 Type A 101 TACCTCAAATTCATGGTCAAAATAAAGGTGATCAGTCATTCGAAGAAGAT 150 J2385 I I I I I I I I I II I I I I II I 11 I III 11111*1 I MM I I I II I l +l I I I I I 2450 TACCTCAAATTCATGGTCAAAATAAAGGTAATCAGTCATTCGAGGAAGAT 2499 Type A 151 ACAGAGAAAGACAAGCCTAAATATGAACATGGTGGTAATATCATTGATAT 200 J2385 M l l l + U IIIII I + M II I + l + IM M + IM M I llll l 2500 ACAGAAAAAGACAAACCTAAGTATGAACATGGCGGTAACATCATTGATAT 2549 Type A 201 CGACTTCGACAGCGTGCCACATATTCATGGATTCAATAAGCACACTGAAA 250 J2385 I I I I I M I I I I l+l I I 1 I 1 I I I I I I I l+l I I I I I I I I I 1 II ! 11 I 11 I I I 2550 CGACTTCGACAGTGTGCCACATATTCACGGATTCAATAAGCACACTGAAA 2599 Type A 251 TTATTGAAGAAGATACAAACAAAGATAAACCAAATTATCAATTCGGTGGA 300 J2385 11 I 1 I 11 I I 1 I I I I I 1 I 1 l+l I I I I II I I I I I I I I 1 I I M I I 11 I I I I 2600 TTATTGAAGAAGATACAAATAAAGATAAACCAAGTTATCAATTCGGTGGA 2649 Type A 301 CACAATAGTGTTGACTTTGAAGAAGATACACTTCCACAAGTAAGTGGTCA 350 J2385 MMI M MMMMMIMMMMMUMMM*!MliI l + M + ll 2650 CACAATAGTGTTGACTTTGAAGAAGATACACTTCCAAAAGTAAGCGGCCA 2699 Type A 351 TAATGAAGGTCAACAAACGATTGAAGAAGATACAAC GC 388 J2385 * 1 I I I I I I I I 1 I I I I I I I I 1 I I I I I I I I I 1 I 1 I M I I I 2700 AAATGAAGGTCAACAAACGATTGAAGAAGATACAACACCTCCAATCGTGC 2749 Type 389 CGCCAACACCACCAACACCAGAAGTACCAAGTGAGCCGGAAACACCAACA 438 J2385 I +1 I I I I +1 I I I I+1 I I I I 1 I I I I 111 I 11 I 1 I M 1 I I I 1 I I i M I 11 I + 2750 CACCAACGCCACCGACACCAGAAGTACCAAGTGAGCCGGAAACACCAACG 2799 Type 439 CCACCGACACCAGAAGTACCAAGTGAGCCGGAAACACCAACACCGCCAAC 488 J2385 MII I+l I IIll M I + ll + l l 2800 CCACCAACACCAGAAGTACCAAGTGAGCCGGAAACACCAACACCACCGAC 2849 Type 489 ACCAGAGGTACCAAGTGAGCCGGAAACACCAACACC 524 J2385 I I I I I l+l l + l l + l I I I 1 I H + l II I l + l I I i I I I I 2850 ACCAGAAGTGCCGAGTGAGCCAGAAACTCCAACACC 2885 Type A + Silent Mutation * Change o : Amino Acid Table 2 A comparison of the derived amino acid sequences of die Fibronectin Binding Regions of die Fibronectin Binding Proteins of Staphylococcus aureus (as published) and Staphylococcus aureus J2385. The Staphylococcus aureus sequence a comprises amino acid residues 709-886 (as in Signas et al. loc. cit.) J2385 GQNSGNQSFE EDTEEOKPKY EQGGNIVDID FDSVPQIHGQ NKGDQSFEED Stapha GQNSGNQSFE EDTEEDKPKY EQGGNIVDID FDSVPQIHGQ NKGNQSFEED Staphb GQNSGNQSFE EDTEEDKPKY EQGGNIVDID FDSVPQIHGQ NNGNQSFEED 51 —> D3 100 J2385 TEKDKPKYEH GGNIIDIDFD SVPHIHGFNK HTEIIEEDTN KDKPNYQFGG Stapha TEKDKPKYEH GGNIIDIDFD SVPHIHGFNK HTEIIEEDTN KDKPSYQFGG Staphb TEKDKPKYEQ GGNIIDIDFD SVPHIHGFNK HTEIIEEDTN KDKPNYQFGG 101 — > D4 — > WR1 146 J2385 HNSVDFEEDT LPQVSGHNEG QQTIEEDTT . . . . PPTPPTP EVPSEPETPT Stapha HNSVDFEEDT LPKVSGQNEG QQTIEEDTTP PIVPPTPPTP EVPSEPETPT Staphb HNSVDFEEDT LPQVSGHNEG QQTIEEDTTP PIVPPTPPTP EVPSEPETPT 174 J2385 PPTPEVPSEP ETPTPPTPEV PSEPETPT Stapha PPTPEVPSEP ETPTPPTPEV PSEPETPT Staphb PPTPEVPSEP ETPTPPTPEV PTEP Figure Fig.l. is a diagrammatic representation of plasmid pBROC413. Bla indicates die ampicillin resistance gene, φlO the T7 RNA polymerase promoter and rbs die ribosome binding site. Arrows for φlO and bla give the direction of transcription. The polylinker site has been indicated. The plasmid is not drawn to scale and die size is approximate. Sequence Listing - Key SEQ ID NOrl FIB 1 GGGAATTCAT ATGGGCCAAA ATAGCGGTAA CCAGTC SEQIDNO:2 FIB2 GCGGATCCTT ACGTTGGTGG CACGATTGGA GGTG SEQ ID NO:3 oligonucleotide (A) CGGAATTCGT CAACAAACGA TTGAAGAAGA TACAACGACG TAAGATCTGG ATCCGCATGC GAATTCCG SEQ ID NO:4 oligonucleotide (B) CGGAATTCGT CAACAAACGA TTGAAGAAGA TACAACGCCG TAAGATCTGG ATCCGCATGC GAATTCCG SEQIDNO:5 GGCCAAAATA GCGGTAACCA GTCATTCGAG GAAGACACAG AAGAAGATAA ACCTAAATAT GAACAAGGTG GCAATATCGT AGATATCGAT TTCGATAGTG TACCTCAAAT TCATGGTCAA AATAAAGGTG ATCAGTCATT CGAAGAAGAT ACAGAGAAAG ACAAGCCTAA ATATGAACAT GGTGGTAATA TCATTGATAT CGACTTCGAC AGCGTGCCAC ATATTCATGG ATTCAATAAG CACACTGAAA TTATTGAAGA AGATACAAAC AAAGATAAAC CAAATTATCA ATTCGGTGGA CACAATAGTG TTGACTTTGA AGAAGATACA CTTCCACAAG TAAGTGGTCA TAATGAAGGT CAACAAACGA TTGAAGAAGA TACAACGCCG CCAACACCAC CAACACCAGA AGTACCAAGT GAGCCGGAAA CACCAACACC ACCGACACCA GAAGTACCAA GTGAGCCGGA AACACCAACA CCGCCAACAC CAGAGGTACC AAGTGAGCCG GAAACACCAA CACCTCCAAT CGTGCCACCA ACGTAA - SEQIDNO:6 D1-D4(709-886) GQNSGNQSFE EDTEEDKPKY EQGGNIVDID FDSVPQIHGQ NKGDQSFEED TEKDKPKYEH GGNIIDIDFD SVPHIHGFNK HTEIIEEDTN KDKPNYQFGG HNSVDFEEDT LPQVSGHNEG QQTIEEDTTP PTPPTPEVPS EPETPTPPTP EVPSEPETPT PPTPEVPSEP ETPTPPIVPP T SEQIDNO:7 D1-D4(709-838(P838T)) (Example7) GQNSGNQSFE EDTEEDKPKY EQGGNIVDID FDSVPQIHGQ NKGDQSFEED TEKDKPKYEH GGNIIDIDFD SVPHIHGFNK HTEIIEEDTN KDKPNYQFGG HNSVDFEEDT LPQVSGHNEG QQTIEEDTTT SEQIDNO:8 D1-D4(709-838) GQNSGNQSFE EDTEEDKPKY EQGGNIVDID FDSVPQIHGQ NKGDQSFEED TEKDKPKYEH GGNIIDIDFD SVPHIHGFNK HTEIIEEDTN KDKPNYQFGG HNSVDFEEDT LPQVSGHNEG QQTIEEDTTP SEQIDNO:9 J2385DNA(Table 1) GGCCAAAATA GCGGTAACCA GTCATTCGAG GAAGACACAG AAGAAGATAA ACCTAAATAT GAACAAGGTG GCAATATCGT AGATATCGAT TTCGATAGTG TACCTCAAAT TCATGGTCAA AATAAAGGTG ATCAGTCATT CGAAGAAGAT ACAGAGAAAG ACAAGCCTAA ATATGAACAT GGTGGTAATA TCATTGATAT CGACTTCGAC AGCGTGCCAC ATATTCATGG ATTCAATAAG CACACTGAAA TTATTGAAGA AGATACAAAC AAAGATAAAC CAAATTATCA ATTCGGTGGA CACAATAGTG TTGACTTTGA AGAAGATACA CTTCCACAAG TAAGTGGTCA TAATGAAGGT CAACAAACGA TTGAAGAAGA TACAACGCCG CCAACACCAC CAACACCAGA AGTACCAAGT GAGCCGGAAA CACCAACACC ACCGACACCA GAAGTACCAA GTGAGCCGGA AACACCAACA CCGCCAACAC CAGAGGTACC AAGTGAGCCG GAAACACCAA CACC SEQIDNO:10 J2385(Table2) GQNSGNQSFE EDTEEDKPKY EQGGNIVDID FDSVPQIHGQ NKGDQSFEED TEKDKPKYEH GGNIIDIDFD SVPHIHGFNK HTEIIEEDTN KDKPNYQFGG HNSVDFEEDT LPQVSGHNEG QQTIEEDTTP PTPPTPEVPS EPETPTPPTP EVPSEPETPT PPTPEVPSEP ETPT IntematioMl Application No: PCT/ MICROORGANISMS Optional Shoot In connection orrtti microorganism referred to on page- 31 . of the deecrtptlon > A. IDENTIFICATION or DEPOSIT • Further deooett* at* Mantffled on «n oddttlonol ahoet ~~ • Homo ot depoaltary Inetttutlon « The National Collection of Industrial and Marine Bacteria Ltd AddrOM ot depoenary Inatftutlon (Including pootal cod* and country) « 23 St . Machar Driv.e Aberdeen AB2 1RY Date el deposit • Acc«— low Number * 8 December 1992 NCIMB 40532 S. AOOITIONAL INDICATION* ' (leave Monk H not applicable). Thla Information la continued on a aaparaw attached ahaot ~~ In respect of those designations in which a European or Australian Patent is sought or in any other states having equivalent provisions, a sample of the deposited micro-organism will be made available until the publication of the mention of the grant of the patent or until the date on which the application has been refused or withdrawn, only by the issue of such a sample to an expert nominated by the person requesting the sample. C. DESIONATED STATE* FOR WHICH INDICATION* AM MAOI • (H the Indication* ara not tor all da*lgn*ι*d State*) ALL O. SEPARATE FURNISHINS OF INDICATIONS • (leave Mank M not applicable) The indication* hated below will be aubmltted to the International Bureau later • (Specify the general nature ot the Indication* e.g., " Accewton Number ol Oepoalt ") t. | * yf * Thι« atieet w.t received with the international application when filed (to be checked by the receiving Office) [ I The dele ot receipt (from the applicant) by the International Bureau *• wa* - - - — - (Authoriied Officer)

Claims: Claims 1. An isolated D 1 -D4 polypeptide from a Staphylococcus aureus Fbp. 2. A polypeptide according to claim 1 consisting of the entire Dl, D2, D3 and D4 regions optionally terminating in PIVP and optionally from one to five wall regions (WR) of Staphylococcus aureus Fbp, in sequence. 3. A polypeptide according to claim 2 containing up to three wall regions. 4. A polypeptide according to claim 3 consisting of residues corresponding to residues G709 to T886 plus PPIVPPT, G709 to P838 or G709 to P838 (P838→T) of Staphylococcus aureus FbpA. 5. A polypeptide according to any preceding claim wherein the Fbp is from S. aureus J2385 characterised by the DNA sequence SEQ ID NO:5. 6. A polypeptide according to claim 1 having the sequence SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8. 7. Isolated nucleic acid encoding the polypeptide of any one of claims 1 to 6. 8. DNA of SEQ ID NO:5. 9. A recombinant vector comprising the isolated nucleic acid of claim 7 or 8. 10. A host cell transformed with the vector of claim 9. 11. A method of preparing the polypeptide of any one of claims 1 to 6 comprising expressing DNA encoding said polypeptide and recovering the expression product. 12. A monoclonal antibody (Mab) that binds to one or more epitopes present on a matrix binding protein. 13. A monoclonal antibody according to claim 12, in which the matrix binding protein is a cell surface protein of Staphylococcus. 14. A monoclonal antibody according to claim 13, in which the matrix binding protein is a fibronectin binding protein (Fbp). 15. A monoclonal antibody according to claim 14, which binds to an epitope present in the D1-D4 region of the Fbp. 16. A monoclonal antibody according to claim 13, 14 or 15, in which the bacterium is a strain of S. aureus or S. epidermidis. 17. A monoclonal antibody according to claim 12, obtainable using an Fbp, or the D1-D4 region of an Fbp, or polypeptide that is an immunologically or antigenically equivalent derivative of the Fbp or D1.D4 region, as antigen. 18. A monoclonal antibody according to claim 12, obtainable using Fbp present on S. aureus J2385 (NCIMB 40532), or a polypeptide that is the D1.D4 region of the Fbp, as antigen. 19. A monoclonal antibody according to any one of claims 12 to 18, which has been humanised. 20. A polypeptide which is a fragment of a monoclonal antibody according to any one of claims 12 to 19 that comprises the binding region of the Mab. 21. A polypeptide that is an Fbp present on 5. aureus J2385 (NCIMB 40532) having a D1-D4 region with the amino acid sequence of SEQ ID NO: 6. 22. A polypeptide that is a derivative of a polypeptide according to claim 6 or 21 and has equivalent immunological or antigenic activity. 23. A process for preparation of monoclonal antibodies in which an animal is immunized with a polypeptide according to any one of claims 1 to 6, 21 or 22, antibody producing cells of the animal are fused with cells of a continuous cell line, and the hybridoma cells are cloned and screened to select clones producing antibodies according to any one of claims 12 to 18. 24. A hybridoma cell line that secretes an antibody according to any one of claims 12 to 18. 25. The plasmid pBROC 520 or pBROC 533. 26. A method of purifying a polypeptide as defined in any one of claims 1 to 6 or 20 to 22 from a sample, comprising immobilizing monoclonal antibodies capable of specifically binding said polypeptide on a substrate, contacting the sample with the immobilised monoclonal antibodies under suitable conditions such that the polypeptide binds to said antibodies, separating unbound sample and eluting the polypeptide from the immobilised monoclonal antibodies. 27. Use of a monoclonal antibody as defined in any one of claims 12 to 19 for the qualitative or quantitative determination of a polypeptide as defined in any one of claims 1 to 6 or 20 to 22. 28. A test kit for the determination of a polypeptide as defined in any one of claims 1 to 6 or 20 to 22 comprising a monoclonal antibody according to any one of claims 12 to 19 and optionally other monoclonal or polyclonal antibodies and / or adjuncts in a suitable package. 29. A pharmaceutical composition comprising a monoclonal antibody according to any one of claims 12 to 19 or a polypeptide according to any one of claims 1 to 6 or 20 and a pharmaceutically acceptable carrier. 30. Use of a polypeptide according to any one of claims 1 to 6 or 20 or a Mab according to any one of claims 12 to 19 in the manufacture of a medicament for use in the prevention of adhesion of bacteria to extracellular matrix proteins on in¬ dwelling devices or to extracellular matrix proteins in wounds. 31. A method of preventing adhesion of bacteria to extracellular matrix proteins present on indwelling devices which comprises treating a patient before, during or after surgery to install the device with an effective amount of a polypeptide according to any one of claims 1 to 6 or 20 or a Mab according to any one of claims 12 to 19. 32. A method of preventing adhesion of bacteria to extracellular matrix proteins present in wounds which comprises treating a wounded patient with an effective amount of a polypeptide according to any one of claims 1 to 6 or 20 or a Mab according to any one of claims 12 to 19. 33. A use or method according to claim 30, 31 or 32 wherein the bacteria are gram positive bacteria.

Inventors: BURNHAM MARTIN KARL RUSSEL, CHOPRA IAN, CRITCHLEY IAN ALFRED, KNOWLES DAVID JUSTIN CHARLES, BURNHAM, MARTIN, KARL, RUSSEL, CHOPRA, IAN, CRITCHLEY, IAN, ALFRED, KNOWLES, DAVID, JUSTIN, CHARLES
Applicants: BURNHAM MARTIN KARL RUSSEL, CHOPRA IAN, CRITCHLEY IAN ALFRED, KNOWLES DAVID JUSTIN CHARLES, SMITHKLINE BEECHAM PLC, BURNHAM, MARTIN, KARL, RUSSEL, CHOPRA, IAN, CRITCHLEY, IAN, ALFRED, KNOWLES, DAVID, JUSTIN, CHARLES
Application year: 1994
Main classification: C12N15
IPC codes: A61K38, A61K39, A61P31, C07K14, C07K16, C12N1, C12N5, C12N15, C12P21, C12R1, G01N33